This application relates to the treatment of neurodegenerative diseases through inhibition of heat shock protein 90 (HSP90).
The HSP90 family of proteins has four recognized members in mammalian cells: Hsp90α and β, Grp94 and Trap-1. Hsp90α and β exist in the cytosol and the nucleus in association with a number of other proteins. Hsp90 in its various forms is the most abundant cellular chaperone, and has been shown in experimental systems to be required for ATP-dependent refolding of denatured or “unfolded” proteins. It has therefore been proposed to function as part of the cellular defense against stress. When cells are exposed to heat or other environmental stresses, the aggregation of unfolded proteins is prevented by pathways that catalyze their refolding or degradation. This process depends on the association of the unfolded protein in an ordered fashion with multiple chaperones (Hsp 60, 90 and 70 and p23), forming a “refoldosome” and ultimately the ATP-dependent release of the chaperones from the refolded protein.
Hsp90 may also play a role in maintaining the stability and function of mutated proteins. It seems to be required for expression of mutated p53 and v-src to a much greater extent than for their wild-type counterparts. It has been suggested that this occurs as a result of Hsp90-mediated suppression of the phenotypes of mutations that lead to protein unfolding.
Hsp90 is also necessary to the conformational maturation of several key proteins involved in the growth response of the cell to extracellular factors. These include the steroid receptors as well as certain transmembrane kinases (i.e., Raf serine kinase, v-src and Her2). The mechanism whereby Hsp90 affects these proteins is not fully understood, but appears to be similar to its role in protein refolding. In the case of the progesterone receptor, it has been shown that binding and release of Hsp90 from the receptor occurs in a cyclic fashion in concert with release of other chaperones and immunophilins and is required for high affinity binding of the steroid to the receptor. Thus, Hsp90 could function as a physiologic regulator of signaling pathways, even in the absence of stress.
Hsp90 has been shown to be overexpressed in multiple tumor types and as a function of oncogenic transformation. Whether it plays a necessary role in maintaining transformation is unknown, but it could have at least three functions in this regard. Cancer cells grow in an environment of hypoxia, low pH and low nutrient concentration. They also rapidly adapt to or are selected to become resistant to radiation and cytotoxic chemotherapeutic agents. Thus, the general role of Hsp90 in maintaining the stability of proteins under stress may be necessary for cell viability under these conditions. Secondly, cancer cells harbor mutated oncogenic proteins. Some of these are gain-of-function mutations which are necessary for the transformed phenotype. Hsp90 may be required for maintaining the folded, functionally-active conformation of these proteins. Thirdly, activation of signaling pathways mediated by steroid receptors, Raf and other Hsp90 targets is necessary for the growth and survival of many tumors which thus probably also require functional Hsp90.
Neurodegeneration, similar to cancer, is likely not the result of a single dysregulatory event, but rather a several-step process involving environmental, epigenetic and genetic events that lead to creation of a complex transformed phenotype manifested by abnormal expression, post-translational modification and processing of certain proteins. The functional maintenance of these dysregulated proteins in neurons may require, analogously to the cancer afflicted cell, the regulatory mechanism of molecular chaperones to evolve along with the transforming process.
In the context of neurodegenerative diseases, Hsp90 may play two roles. First, aberrantly activated kinases (such as cdk5/p35, gsk3beta) in neurodegenerative diseases may require Hsp90 for functioning. Thus, Hsp90 inhibition may restore damaged signaling networks in the diseased brain by alleviating aberrant phosphorylation, leading to reduced aberrant protein aggregation, and elimination or reduction of aggregates and of their associated toxicity. Second, pathogenic mutants (such as of APP or presenilins in AD or mtau in FTDP-17 or mutant androgen receptor in bulbar muscular atrophy) may require Hsp90 for correct folding and functioning, thus Hsp90 inhibition may lead to the elimination of these proteins and result in reduction of aggregates and consequent plaque or tangle formation.
Most neurodegenerative diseases are probably characterized by both mutants and aberrant signaling, and Hsp90 can play a role with respect to pathogenic mutants as well. Tau mutations cause autosomal dominant frontal temporal dementia. Pathologies linked to mutations of the androgen receptor include the complete androgen insensitivity syndrome (CAIS) and the spinal and bulbar muscular atrophy (SBMA or Kennedy's disease). (4) Mutations in the presenilin genes are the major cause of familial AD. Analysis of conditional knockout mice has shown that inactivation of presenilins results in progressive memory impairment and age-dependent neurodegeneration, suggesting that reduced presenilin activity might represent an important pathogenic mechanism. Presenilins positively regulate the transcription of cAMP response element (CRE)-containing genes, some of which are known to be important for memory formation and neuronal survival. (5) Alzheimer's Disease (AD) is characterized both by NFTs (tau aggregates) and plaques (Aβ deposits). In Alzheimer's disease, mutations in amyloid precursor protein or in the presenilins cause autosomal dominant disease. These are the substrate and proteases responsible for the production of the deposited peptide A. Prion mutations cause Gerstmann Straussler syndrome and hereditary Creutzfeldt-Jakob disease, alpha-synuclein mutations cause autosomal dominant Parkinson's disease. In these cases, the pathogenic mutation is in the protein that is deposited in the diseased tissue and the whole protein is deposited. Huntington D results from a mutant huntingtin. (9) Thus, in all the cases, the mutations lead to the disease by a mechanism that involves the deposition process.
These characteristics of Hsp90 make it a viable target for therapeutic agents. HSP90 family members possess a unique pocket in their N-terminal region that is specific to and conserved among all Hsp90s from bacteria to mammals, but which is not present in other molecular chaperones. The endogenous ligand for this pocket is not known, but it binds ATP and ADP with low affinity and has weak ATPase activity. The ansamycin antibiotics geldanamycin (GM) and herbimycin (HA) have been shown to bind to this conserved pocket, and this binding affinity has been shown for all members of the HSP90 family. International Patent Publication No. WO98/51702 discloses the use of ansamycin antibiotics coupled to a targeting moiety to provide targeted delivery of the ansamycin leading to the degradation of proteins in and death of the targeted cells. International Patent Publication No. WO00/61578 relates to bifunctional molecules having two moieties which interact with the chaperone protein Hsp90, including in particular homo- and heterodimers of ansamycin antibiotics. These bifunctional molecules act to promote degradation and/or inhibition of HER-family tyrosine kinases and are effective for treatment of cancers which overexpress Her-kinases.
Exemplary small molecule therapeutics that bind to the same binding pocket of Hsp90 as ATP and the ansamycin antibiotics are disclosed in PCT Publication No. WO02/36075, PCT Application No. PCT/US06/03676 and US Patent Publications 2005-0113339, 2005-0004026, 2005-0049263, 2005-0256183, 2005-0119292, 2005-0113340 and 2005-0107343, all of which are incorporated herein by reference.
In aged organisms, chaperone overload leads to a significant decrease in the robustness of cellular networks shifting their function towards a more stochastic behavior. Unbalanced chaperone requirement and chaperone capacity helps the accumulation of misfolded and aggregated proteins especially in the nervous system, due to the very limited proliferation potential of neurons. In addition, damaged signaling networks lose their original stringency, and irregular protein phosphorylation occurs. An appealing approach to alleviating and reversing such damaging effects is by modulating Hsp90 activity Inhibitors of Hsp90 activity release HSF1 from Hsp90 complexes and correct the defective regulation of HSF1 activity after heat stress leading to an increase in cellular levels of chaperones, such as Hsp70 and Hsp40. Overexpression of these chaperones has been shown to represent a general way of reinstating proper folding and alleviating misfolded proteins' toxic effects. In addition to their effects on reinstating correct folding, Hsp90 inhibitors may regulate proteins involved in signaling networks of diseased neurons.
The usefulness of Hsp90 inhibitors as clinical agents in the treatment of neurodegenerative diseases, however, will depend on whether their effects occur at concentrations of drug that are tolerable to the patient and on whether the drugs can be administered in such a fashion so as to achieve these concentrations in the brain. Unfortunately, known Hsp90 inhibitors such as geldanamycin and 17AAG, its derivative in Phase I clinical trial for cancer, and the unrelated compound radicicol have significant limitations. They are poorly soluble, difficult to formulate and do not cross the blood-brain barrier. Thus, in order to realize the potential for treatment of neurodegenerative diseases, therapeutic agents that inhibit Hsp90, and that have sufficient solubility and the ability to cross the blood-brain barrier or otherwise be delivered to the brain are needed.